Organic solvent pretreatment of biomass to enhance enzymatic saccharification

ABSTRACT

Biomass is pretreated using an organic solvent solution under alkaline conditions in the presence of one of more sulfide (hydrosulfide) salt and optionally one or more additional nucleophile to fragment and extract lignin. Pretreated biomass is further hydrolyzed with a saccharification enzyme consortium. Fermentable sugars released by saccharification may be utilized for the production of target chemicals by fermentation.

The application claims the benefit of U.S. Provisional Application No.61/139,163, filed Dec. 19, 2008, the disclosure of which is herebyincorporated in its entirety.

FIELD OF THE INVENTION

Methods for producing readily saccharifiable carbohydrate-enrichedlignocellulosic biomass are provided and disclosed. Specifically,pretreated biomass is prepared through simultaneous fragmentation andselective extraction of lignin in an organic solvent solution atelevated temperatures in the presence of a sulfide salt such as ammoniumsulfide at an alkaline pH. Optionally, one or more alkylamine andvarious nucleophiles may be added to the biomass pretreatment solution.The remaining carbohydrate-enriched solids in the pretreated biomass maythen be subjected to enzymatic saccharification to obtain fermentablesugars, which may be subjected to further processing for the productionof target products.

BACKGROUND OF THE INVENTION

Cellulosic and lignocellulosic feedstocks and wastes, such asagricultural residues, wood, forestry wastes, sludge from papermanufacture, and municipal and industrial solid wastes, provide apotentially large renewable feedstock for the production of chemicals,plastics, fuels and feeds. Cellulosic and lignocellulosic feedstocks andwastes, composed of cellulose, hemicellulose, pectins and of lignin aregenerally treated by a variety of chemical, mechanical and enzymaticmeans to release primarily hexose and pentose sugars, which can then befermented to useful products.

Pretreatment methods are often used to make the polysaccharides oflignocellulosic biomass more readily accessible to cellulolytic enzymes.One of the major impediments to cellulolytic enzyme digest is thepresence of lignin, a barrier that limits the access of the enzymes totheir substrates, and a surface to which the enzymes bindnon-productively. Because of the significant costs associated withenzymatic saccharification, it is desirable to minimize the enzymeloading by either inactivation of the lignin to enzyme adsorption or itsoutright extraction. Another challenge is the inaccessibility of thecellulose to enzymatic hydrolysis either because of its protection byhemicellulose and lignin or by its crystallinity. Pretreatment methodsthat attempt to overcome these challenges include: steam explosion, hotwater, dilute acid, ammonia fiber explosion, alkaline hydrolysis(including ammonia recycled percolation), oxidative delignification andorganosolv.

Organosolv methods, as previously practiced for the treatment oflignocellulose biomass, for either the production of pulp or forbiofuels applications, while generally successful in lignin removal,have suffered from poor sugar recoveries, particularly of xylose. Forexample, the use of slightly acidic ethanol-water mixtures (e.g., EtOH42 weight %) at elevated temperature to remove lignin fromlignocellulosic biomass (Kleinert, T. N., Tappi 57: 99-102, 1974)resulted in substantial loss of carbohydrate. Dilute acid hydrolysis at95° C. followed by organic solvent extraction and enzymaticsaccharification (Lee, Y-H. et al., Biotech. Bioeng., 29: 572-581, 1987)resulted in substantial loss of hemicellulose during hydrolysis,additional carbohydrate loss upon organic solvent extraction and pooryield (˜50% of total carbohydrate) upon enzymatic saccharification ofresidue. Use of aqueous organic solvent containing ammonia at elevatedtemperatures to treat lignocellulosic biomass (Park J.-K. and Phillips,J. A., Chem. Eng. Comm., 65: 187-205, 1988) required the use of a highliquid to solids ratio in pretreatment and resulted in substantial lossof hemicellulose and poor enzymatic saccharification of cellulose.Treatment of biomass with gaseous water and methylamine followed byextraction with organic solvent and then extraction with water, requiredthree steps and resulted in a substantial loss of carbohydrate(Siegfried, P. and Götz, R., Chem. Eng. Technol., 15: 213-217, 1992).Treatment with polyamines or ethylamine in water-aliphatic alcoholmixtures plus catalyst at elevated temperature required highliquid/solids ratio and low concentrations of alcohol led to poor sugarrecovery, particularly of xylan (U.S. Pat. No. 4,597,830A).Thioglycolate in aqueous alkaline solution used to treat lignocellulosicbiomass at elevated temperature, followed by a hot water wash requireduse of alkali-metal or alkaline-earth hydroxides. This method requiresthe costly disposal of inorganic ions, high weight % thioglycolate, anduse of large volumes of water (U.S. Pat. No. 3,490,993). Treatment withorganic solvent-water mixtures in the presence of sulfide/bisulfide atelevated temperatures required a high solvent/solids ratio and elevatedsulfur content and resulted in a substantial loss of carbohydrate, (U.S.Pat. No. 4,329,200A).

Additional shortcomings of previously applied methods include, separatehexose and pentose streams (e.g. dilute acid), inadequate ligninextraction or lack of separation of extracted lignin frompolysaccharide, particularly in those feedstocks with high lignincontent (e.g., sugar cane bagasse, softwoods), need to dispose of wasteproducts (e.g., salts formed upon neutralization of acid or base), andpoor recoveries of carbohydrate due to breakdown or loss in wash steps.Other problems include the high cost of energy, capital equipment, andpretreatment catalyst recovery, and incompatibility withsaccharification enzymes.

One of the major challenges of biomass pretreatment is to maximize theextraction or chemical neutralization (with respect to non-productivebinding of cellulolytic enzymes) of the lignin while minimizing the lossof carbohydrate (cellulose plus hemicellulose) via low-cost, efficientprocesses. The higher the selectivity, the higher the overall yield ofmonomeric sugars following combined pretreatment and enzymaticsaccharification.

There is therefore a need to develop a single step process usingsubstantially lower concentrations of sulfur and recyclable base in theform of ammonia or alkylamines as opposed to the use of alkali metalhydroxides which are not amenable to either recycle or disposal. Thecurrent disclosure addresses this need. In this disclosure, a sulfidesalt such as ammonium sulfide, in an organic solvent-mediated processand at alkaline pH, and optionally various nucleophiles followed byselective extraction of lignin at elevated temperatures is used.Surprisingly, this cost-effective process resulted in significantlyimproved lignin fragmentation and extraction and high carbohydrateretention.

SUMMARY OF THE INVENTION

The present invention provides methods for producing readilysaccharifiable carbohydrate-enriched biomass and for selectivelyextracting lignin from lignocellulosic biomass while nearlyquantitatively retaining carbohydrate. The methods include treatinglignocellulosic biomass with an organic solvent, such as EtOH in H₂O,and one or more sulfide (hydrosulfide) salt under alkaline conditions atelevated temperatures in a single step. In another embodiment thesolvent solution further comprises one or more alkylamines. Followingpretreatment, the biomass may be further treated with a saccharificationenzyme consortium to produce fermentable sugars. These sugars may besubjected to further processing for the production of target products.

-   -   Accordingly, the invention provides a method for producing        carbohydrate-enriched biomass comprising:        -   (a) providing lignocellulosic biomass comprising lignin;        -   (b) suspending the biomass of (a) in an organic solvent            solution comprising water and one or more sulfide salt under            alkaline conditions whereby a biomass-solvent suspension is            formed;        -   (c) heating the biomass-solvent suspension to a temperature            of about 100° C. to about 220° C. for about 5 minutes to            about 5 hours whereby lignin is fragmented and is dissolved            in the suspension; and        -   (d) filtering free liquid whereby the dissolved lignin is            removed and whereby readily carbohydrate-enriched biomass is            produced.    -   In another embodiment the invention provides a method of        simultaneous fragmentation and selective extraction of lignin        from lignocellulosic biomass comprising:        -   (a) providing:            -   1) an amount of lignocellulosic biomass;            -   2) a multi-component organic solvent solution comprising                from about 40% to about 70% ethanol in water;            -   3) one or more sulfide salt(s); and            -   4) one or more alkylamine(s) under alkaline conditions;        -   (b) contacting said biomass with the multi-component solvent            solution of (a) whereby a solvent-biomass mixture is            produced; (c) placing the solvent-biomass mixture in a            sealed pressure vessel whereby the mixture of (b) is heated            from about 100° C. to about 220° C. for about 5 minutes to            about 5 hours whereby lignin is fragmented and dissolved in            the solvent;        -   (d) removing the dissolved lignin of (c) by filtration; and        -   (e) washing the residual biomass with organic solvent,            whereby substantially lignin-free biomass is produced.

Particularly suitable one or more sulfide (hydrosulfide) salt includesammonium sulfide (hydrosulfide). In another aspect, one or morealkylamine or an amount of ammonia is present in the solvent solution.

Particularly suitable feedstocks for use in the methods of the inventioninclude but are not limited to switchgrass, waste paper, sludge frompaper manufacture, corn fiber, corn cobs, corn husks, corn stover,grasses, wheat, wheat straw, hay, barley, barley straw, rice straw,sugar cane bagasse, sugar cane straw, yellow poplar, sorghum, soy,components obtained from processing of grains, trees, branches, roots,leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits,flowers, animal manure and combinations thereof.

Particularly suitable alkylamines include those selected from the groupconsisting of R—NH₂, R₂—NH, R₃N, (H₂N—R—NH₂), (H₂N—R(NH₂)₂), (HO—R—NH₂),((HO)₂—R—NH₂), (HO—R—(NH₂)₂), (HS—R—NH₂), ((HS)₂—R—NH₂), (HS—R—(NH₂)₂)and (H₂N—R(OH)(SH) and combinations thereof, wherein R is independentlya monovalent, divalent or trivalent 1-6 carbon alkane, alkene or alkyne,linear, cyclic or branched.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1—FIG. 1 shows the UV absorbance spectra of filtrates (diluted1:5000 with 70% EtOH in H₂O (v/v)) following pretreatment at 187° C. for1 hour in 70% EtOH in H₂O (v/v) plus 14% methylamine (w/w biomass) withor without 2% or 6% (NH₄)₂S.

DETAILED DESCRIPTION OF THE INVENTION

Applicants specifically incorporate the entire content of all citedreferences in this disclosure. Unless stated otherwise, all percentages,parts, ratios, etc., are by weight. Trademarks are shown in upper case.Further, when an amount, concentration, or other value or parameter isgiven as either a range, preferred range or a list of upper preferablevalues and lower preferable values, this is to be understood asspecifically disclosing all ranges formed from any pair of any upperrange limit or preferred value and any lower range limit or preferredvalue, regardless of whether ranges are separately disclosed. Where arange of numerical values is recited herein, unless otherwise stated,the range is intended to include the endpoints thereof, and all integersand fractions within the range. It is not intended that the scope of theinvention be limited to the specific values recited when defining arange.

The present invention provides a process for the treatment of biomass inorder to produce readily saccharifiable carbohydrate-enriched biomass toenhance the subsequent enzymatic saccharification step. A processinvolving a pretreatment step wherein lignin is simultaneouslyfragmented and extracted using an organic solvent under alkalineconditions at elevated temperatures in the presence of one or moresulfide salt is employed. Additional nucleophiles may be employed forfurther benefit. The treated biomass is then filtered and washed toremove solubilized lignin, acetic acid, acetamides, alkylamides andexcess reagent and then digested with a saccharification enzymeconsortium to produce readily fermentable sugars. The sugars may then befurther processed to one or more target product. The removed lignin mayalso be further processed and utilized for other purposes (such asburning for energy) to increase efficiency.

DEFINITIONS

The following definitions are used in this disclosure:

“Room temperature” and “ambient” when used in reference to temperaturerefer to any temperature from about 15° C. to about 25° C.

“Fermentable sugars” refers to a sugar content primarily comprisingmonosaccharides and some disaccharides that can be used as a carbonsource by a microorganism (and some polysaccharides may be present)) ina fermentation process to produce a target product. “Readily fermentablesugars” means that additional costly processing is not necessary and/orthat a fermentative microorganism can be contacted with the resultingsugars with minimal impediments from inhibitors or other components thatmay adversely affect fermentation.

“Lignocellulosic” refers to material comprising both lignin andcellulose. Lignocellulosic material may also comprise hemicellulose. Inthe processes described herein, lignin is dissolved and substantiallyremoved from the lignocellulosic biomass to produce acarbohydrate-enriched biomass.

“Dissolved lignin” as referred to herein means the lignin that isdissolved in an organic solvent solution.

“AI lignin” refers to acid-insoluble lignin.

“Autohydrolysis” refers to the hydrolysis of biomass in the presence ofsolvent (water or organic solvent plus water) plus heat with no furtheradditions, such as without exogenous acid or base or hydrolytic enzymeaddition.

“Cellulosic” refers to a composition comprising cellulose.

“Target product” refers to a chemical, fuel, or chemical building blockproduced by fermentation. Product is used in a broad sense and includesmolecules such as proteins, including, for example, peptides, enzymesand antibodies. Also contemplated within the definition of targetproduct are ethanol and butanol.

“Dry weight of biomass” refers to the weight of the biomass having allor essentially all water removed. Dry weight is typically measuredaccording to American Society for Testing and Materials (ASTM) StandardE1756-01 (Standard Test Method for Determination of Total Solids inBiomass) or Technical Association of the Pulp and Paper Industry, Inc.(TAPPI) Standard T-412 om-02 (Moisture in Pulp, Paper and Paperboard).

“Selective extraction” means removal of lignin while substantiallyretaining carbohydrates.

“Solvent solution” and “an organic solvent solution”, as used herein, isan organic solvent mixture in water that includes any organic liquidthat dissolves a solid, liquid, or gaseous solute, resulting in asolution. The most suitable solvent solutions for this invention areorganic solvents such as ethanol, methanol, n-propanol, isopropanol,n-butanol, 2-butanol, isobutanol, t-butanol, pentanol and hexanol anddiols with the same number of carbons. They can also include aproticsolvents. The solvent solutions can include additional components inmixture with the solution, e.g, the solvent solution may include one ormore nucleophile.

“Biomass” and “lignocellulosic biomass” as used herein refer to anylignocellulosic material, including cellulosic and hemi-cellulosicmaterial, for example, bioenergy crops, agricultural residues, municipalsolid waste, industrial solid waste, yard waste, wood, forestry wasteand combinations thereof, and as further described below. Biomass has acarbohydrate content that comprises polysaccharides and oligosaccharidesand may also comprise additional components, such as protein and/orlipid.

“Highly conserved” as used herein refers to the carbohydrate content ofthe lignocellulosic material after the processing steps describedherein. In an embodiment of the invention, the highly conservedcarbohydrate content provides for sugar yields after saccharificationthat are substantially similar to theoretical yields with minimal lossof sugar yield from the processes described herein. In an embodiment ofthe invention, highly-conserved with reference to carbohydrate contentrefers to the conservation of greater than or equal to 85% of thebiomass carbohydrate as compared to biomass prior to pretreating asdescribed herein.

“Preprocessing” as used herein refers to processing of lignocellulosicbiomass prior to pretreatment. Preprocessing is any treatment of biomassthat prepares the biomass for pretreatment, such as mechanically millingand/or drying to the appropriate moisture contact.

“Biomass-solvent suspension” refers to a mixture of biomass and solvent.The biomass-solvent solution may comprise additional components such asone or more sulfide salt, one or more alkylamine, etc.

“Saccharification” refers to the production of fermentable sugars fromprimarily polysaccharides by the action of hydrolytic enzymes.Production of fermentable sugars from pretreated biomass occurs byenzymatic saccharification by the action of cellulolytic andhemicellulolytic enzymes.

“Pretreating biomass” or “biomass pretreatment” as used herein refers tosubjecting native or preprocessed biomass to chemical or physicalaction, or any combination thereof, rendering the biomass moresusceptible to enzymatic saccharification or other means of hydrolysisprior to saccharification. For example, the methods claimed herein maybe referred to as pretreatment processes that contribute to renderingbiomass more accessible to hydrolytic enzymes for saccharification.

“Pretreatment filtrate” means the free liquid that is in contact withthe biomass following pretreatment and which is separated by filtration.

“Pretreated Biomass” as used herein refers to native or preprocessedbiomass that has been subjected to chemical or physical action, or anycombination thereof, rendering the biomass more susceptible to enzymaticsaccharification or other means of hydrolysis prior to saccharification.

“Air-drying the filtered biomass” can be performed by allowing thebiomass to dry through equilibration with the air of the ambientatmosphere.

“Readily saccharifiable biomass” means biomass that iscarbohydrate-enriched and made more amenable to hydrolysis bycellulolytic or hemi-cellulolytic enzymes for producing monomeric andoligomeric sugars, i.e., pretreated biomass as described herein.

“Carbohydrate-enriched” as used herein refers to the biomass produced bythe process treatments described herein. In one embodiment the readilysaccharifiable carbohydrate-enriched biomass produced by the processesdescribed herein has a carbohydrate concentration of greater than orequal to 85% of the dried biomass by weight, while having removed 75% orgreater of the starting biomass lignin content based on dry weight.

“Heating the biomass suspension” means subjecting the biomass suspendedin a solvent to a temperature greater than ambient or room temperature.Temperatures relevant to the present pretreatments are from about 100 toabout 220° C., or from about 140 to about 180° C., or any temperaturewithin or approximately these ranges.

“Filtering free liquid under pressure” means removal of unbound liquidthrough filtration, with some pressure difference on opposite faces ofthe filter

“Alkaline” or “under alkaline conditions” means a pH of thebiomass-solvent suspension equal to or greater than the pKas of thenucleophiles present such that these are substantially deprotonated andmore highly reactive than in their protonated states. These nucleophileswould include alkylamines, and ammonia, thiols, polysulfides andhydrosulfide (if present).

“Divalent alkane” means a linear, branched or cyclic alkane with twoopen valences.

For the purposes of this invention, an organic solvent solutioncomprising a sulfide salt refers to the use of compounds comprisingsulfide (S⁼) or polysulfide (S_(n)S⁼, where n is an integer) ions suchas ammonium sulfide, compounds that release sulfides or polysulfidesupon disproportionation of elemental sulfur under alkaline conditions atelevated temperature or in which oxides of sulfur are combusted in thepresence of electron-rich sources such as lignin to produce sulfides (asin the Kraft process). If alkylamines are present in the organosolvsolution, then alkylammonium salts of sulfides are produced that performsimilarly to ammonium sulfide. Inorganic salts of sulfide or polysulfide(e.g. Na₂S or Na₂S_(n)) may also be used in the organic solventsolution.

“Alkylamine” means an alkane containing an —NH₂ group in place of one,two or three H atoms; e.g., monomethylamine, dimethylamine,trimethylamine, ethylamine, isopropyl-amine, ethylhexylamine,cyclohexylamine, and as further defined below.

“Air-dried sample” means a pretreated sample which had been allowed toair-dry at ambient temperature and pressure to the point where itsmoisture content was in equilibrium with that of the ambient air,typically ≧85% dry matter.

“Substantially lignin-free biomass” means a pretreated sample in whichabout ≧75% of the lignin is removed.

“Dry biomass” means biomass with a dry matter content of ≧85%. Methodsfor drying the biomass include exposure at ambient temperature to vacuumor flowing air at atmospheric pressure and or heating in an oven or avacuum oven.

“Multi-component solvent” means a solvent containing organic solvent,water, and reagents capable of chemical attack on the lignin.

“Pressure vessel” is a sealed vessel that may be equipped or not with amechanism for agitation of a biomass/solvent suspension, in which apositive pressure is developed upon heating the lignocellulosic biomass.

“Nucleophile” is a chemical reagent capable of forming a covalent bondwith its reaction partner by contributing both of the bonding electrons.

“Hydrolysate” refers to the liquid in contact with the lignocellulosebiomass which contains the products of hydrolytic reactions acting uponthe biomass (either enzymatic or not), in this case monomeric andoligomeric sugars.

“Organosolv” means a mixture of organic solvent and water which istypically in contact with biomass and in which the lignin or itsfragments are soluble.

“Enzyme consortium” or “saccharification enzyme consortium” is acollection of enzymes, usually secreted by a microorganism, which in thepresent case will typically contain one or more cellulases, xylanases,glycosidases, ligninases and esterases.

“Monomeric sugars” or “simple sugars” consist of a single pentose orhexose unit, e.g., glucose, xylose and arabinose.

“Delignification” is the act of removing lignin from lignocellulosicbiomass. In the context of this application, delignification meansfragmentation and extraction of lignin from the lignocellulosic biomassusing an organic solvent under alkaline conditions at elevatedtemperatures in the presence of alkylamines and optionally variousnucleophiles.

“Fragmentation” is a process in which lignocellulosic biomass is treatedwith organic solvent under alkaline conditions breaking the lignin downinto smaller subunits.

“Selective extraction” is a process by which fragmented lignin isdissolved by treatment with an organic solvent under alkaline conditionsleaving behind the polysaccharide.

“Simultaneous fragmentation and selective extraction” as used hereinrefers to a fragmentation reaction performed in organic solvent suchthat the lignin fragments go into solution as soon as they are releasedfrom the bulk biomass.

Methods for pretreating lignocellulosic biomass to produce readilysaccharifiable biomass are provided. These methods provide economicalprocesses for rendering components of the lignocellulosic biomass moreaccessible or more amenable to enzymatic saccharification. Thepretreatment can be chemical, physical or biological, or any combinationof the foregoing. In this disclosure the pretreatment is performed inthe presence of nucleophiles, specifically in the presence of a sulfidesalt such as ammonium sulfide under alkaline conditions. Additionalnucleophiles may also be present, such as NH₃, one or more alkylamines,sulfide reagents, or combinations thereof. The presence of an organicsolvent and alkaline conditions assists lignin fragmentation and removaland carbohydrate recovery.

In addition, the methods described in the present disclosure minimizethe loss of carbohydrate during the pretreatment process and maximizethe yield of solubilized (monomeric+oligomeric) sugars insaccharification.

As disclosed above the methods described herein include pretreatinglignocellulosic material, with a solvent solution comprising thecomponents described below, to produce a readily saccharifiablecarbohydrate-enriched biomass.

Solvents

The methods described herein include use of an organic solvent forpretreating biomass and specifically for fragmentation and extraction oflignin. Solvents useful in the present methods are frequently referredto in the art as Organosolv (e.g., E. Muurinen (2000) OrganosolvPulping, A review and distillation study related to peroxyacid pulpingThesis, University of Oulu, pp. 314; S. Aziz, K. Sarkanen, Tappi J.,72/73: 169-175, 1989; A. K. Varsheny and D. Patel, J. Sci. Ind. Res.,47: 315-319, 1988; A. A. Shatalov and H. Pereira, BioResources 1: 45-61,2006; T. N. Kleinert, Tappi J., 57: 99-102, 1979; Practice of organosolvtechnology for biofuels, derived from Kleinert, which has advanced tothe pilot scale using EtOH/H₂O has been described (WO 20071051269), andX. Pan, N. Gilkes, J. Kadla, K. Pye, S. Saka, D. Gregg, K. Ehara, D.Xie, D. Lam, and J. Saddler, Biotechnol. Bioeng., 94: 851-861, 2006.While still at lab scale, use of acetone/H₂O is described in U.S. Pat.No. 4,470,851. Further details on pretreatment technologies related touse of solvents and other pretreatments can be found in Wyman et al.,(Bioresource Tech., 96: 1959, 2005); Wyman et al., (Bioresource Tech.,96: 2026, 2005); Hsu, (“Pretreatment of biomass” In Handbook onBioethanol: Production and Utilization, Wyman, Taylor and Francis Eds.,p. 179-212, 1996); and Mosier et al., (Bioresource Tech., 96: 673,2005). Solvents are used herein for pretreating biomass to removelignin. Delignification is typically conducted at temperatures of165-225° C., at liquid to biomass ratios of 4:1 to 20:1, at liquidcompositions of 50% organic solvent (v/v), and at reaction times between0.5-12 hours. A number of mono- and polyhydroxy-alcohols have beentested as solvents. Ethanol, butanol and phenol have been used in thesereactions (Park, J. K., and Phillips, J. A., Chem. Eng. Comm., 65:187-205, 1988).

The organosolv or organic solvent solution pretreatment in the presentmethods may comprise a mixture of water and an organic solvent atselected condition parameters that include temperature, time, pressure,solvent-to-water ratio and solids-to-liquid ratio. The solvent cancomprise, but is not limited to, alcohols and aprotic solvents (solventsthat do not have a hydrogen atom bound to an oxygen as in a hydroxylgroup or a nitrogen as in an amine group or a sulfur as in a thiolgroup, e.g., ketones). The alcohols may include methanol, ethanol,propanol, butanol, pentanol and hexanol and isomers thereof and diolswith the same number of carbon atoms, such as 1,2-ethanediol,1,2-propandiol, 1,3-propanediol, 1,3-hexanediol.

The concentration of the solvent in solution (i.e. water) in the presentinvention is from about 2 to about 90% (v/v), or from about 10% to about85% or from about 20% to about 80% or from about 30% to about 80% ormore preferably from about 40% to about 70% (v/v). Specifically, forpurposes of an embodiment of the methods herein, EtOH in H₂O mixturesfrom about 0%-80% (v/v) ethanol concentrations were examined andsolutions containing 40-70% (v/v) EtOH were found to be most effective.

Sulfide or Polysulfide Salts

According to the present method, ammonium sulfide is added to thealkaline organic solvent mixture in the presence of ammonia oralkylamines increasing lignin fragmentation and extraction, andresulting in an increased accessibility of the carbohydrate-enrichedbiomass to enzymatic saccharification. In the present invention,concentrations of ammonium sulfide from 0.5% to 15% (w/w biomass) couldbe used. More specifically concentrations of 1% to 6% (w/w biomass) aremore useful. Even more specifically concentrations of 2% to 4% (w/wbiomass) would be most useful. Alkylammonium sulfides function similarto ammonium sulfide, with the added advantage that alkylamines arebetter nucleophiles than ammonia. In addition ammonium and alkylammoniumpolysulfides are expected to behave like the sulfides. Inorganic ions ofsulfides and polysulfides also work under organosolv conditions tofragment and extract lignin.

Additional Components of the Solvent Solution

In one embodiment, alkylamines are used for pretreatment of biomassaccording to the present methods as components of the organic solventsolution. Alkylamines are strong bases owing to electron donation to theamine nitrogen by the alkyl chain carbons, and consist of primary amines(R—NH₂), secondary amines (R—N—R′) and tertiary amines where R is analkyl chain. Specifically R could be selected from a group consisting ofa monovalent, divalent or trivalent 1-6 carbon alkane, alkene or alkyne,linear, cyclic or branched. Examples of alkylamines include, mono, di-and tri-methylamine, mono, di- and tri-ethylamine, mono, di- andtri-propylamine, mono, di- and tri-butylamine. Alkylamines includemono-, di- and tri-amines, alcohol amines (HO—R—NH₂), diolamines((HO)₂—R—NH₂), alcohol diamines (HO—R—(NH₂)₂), thiolamines (HS—R—NH₂),dithiolamines ((HS)₂—R—NH₂), thioldiamines (HS—R—(NH₂)₂) and alcoholthiolamines (H₂N—R(OH)(SH) where R is as defined.

Suitable alkylamines for this invention comprise: methylamine (MA),dimethylamine (DMA), trimethylamine (TMA), ethylamine, propylamine, andbutylamine. The more suitable alkylamines for this invention include,but are not limited to MA and DMA. The concentration of the alkylaminesaccording to the present method may be used from about 1% to about 20 wt% of dry biomass. In accordance with the present methods alkylamines,especially MA and DMA, are highly active in a concentration ranges offrom 10 to 14% relative to dry weight of biomass. In this concentrationrange there is sufficient alkylamine to assure that the pH of thesolvent solution remains high and that the concentration of alkylamineis sufficient to assure continued lignin fragmentation as pretreatmentoccurs.

The inorganic base could be used at various concentrations of at leastfrom 0.5% to about 16% (wt % of dry biomass). More suitable are theconcentrations from 1% to 10%. Most suitable are the concentrationsbetween 2% to 8% (wt % of dry biomass).

Lignocellulosic Biomass

The lignocellulosic biomass pretreated herein includes, but is notlimited to, bioenergy crops, agricultural residues, municipal solidwaste, industrial solid waste, sludge from paper manufacture, yardwaste, wood and forestry waste. Examples of biomass include, but are notlimited to corn cobs, crop residues such as corn husks, corn stover,grasses, wheat, wheat straw, barley, barley straw, hay, rice straw,switchgrass, waste paper, sugar cane bagasse, sugar cane straw, yellowpoplar, sorghum, soy, components obtained from milling of grains, trees,branches, roots, leaves, wood chips, sawdust, shrubs and bushes,vegetables, fruits, flowers and animal manure.

In one embodiment, the lignocellulosic biomass includes agriculturalresidues such as corn stover, wheat straw, barley straw, oat straw, ricestraw, canola straw, and soybean stover; grasses such as switchgrass,miscanthus, cord grass, and reed canary grass; fiber process residuessuch as corn fiber, beet pulp, pulp mill fines and rejects and sugarcane bagasse; sugar cane straw and sorghum; forestry wastes such asyellow poplar, aspen wood, other hardwoods, softwood and sawdust; andpost-consumer waste paper products; as well as other crops orsufficiently abundant lignocellulosic material.

In another embodiment, biomass that is useful for the invention includesbiomass that has a relatively high carbohydrate content, is relativelydense, and/or is relatively easy to collect, transport, store and/orhandle.

In another embodiment of the invention, biomass that is useful includescorn cobs, corn stover, sugar cane bagasse, sugar cane straw, yellowpoplar and switchgrass.

The lignocellulosic biomass may be derived from a single source, orbiomass can comprise a mixture derived from more than one source; forexample, biomass could comprise a mixture of corn cobs and corn stover,or a mixture of stems or stalks and leaves.

In the present method, the biomass dry weight is at an initialconcentration of at least about 9% up to about 80% of the weight of thebiomass-solvent suspension during pretreatment. More suitably, the dryweight of biomass is at a concentration of from about 15% to about 70%,15% to about 60%, or about 15% to about 50% of the weight of thebiomass-solvent suspension. The percent of biomass in thebiomass-solvent suspension is kept high to reduce the total volume ofpretreatment material, decreasing the amount of solvent and reagentsrequired and making the process more economical.

The biomass may be used directly as obtained from the source, or may besubjected to some preprocessing, for example, energy may be applied tothe biomass to reduce the size, increase the exposed surface area,and/or increase the accessibility of lignin and of cellulose,hemicellulose, and/or oligosaccharides present in the biomass toorganosolv pretreatment and to saccharification enzymes used,respectively, in the second and third steps of the method. Energy meansuseful for reducing the size, increasing the exposed surface area,and/or increasing the accessibility of the lignin, and the cellulose,hemicellulose, and/or oligosaccharides present in the biomass to theorganosolv pretreatment and to saccharification enzymes include, but arenot limited to, milling, crushing, grinding, shredding, chopping, discrefining, ultrasound, and microwave. This application of energy mayoccur before or during pretreatment, before or during saccharification,or any combination thereof.

Drying prior to pretreatment may occur as well by conventional means,such as exposure at ambient temperature to vacuum or flowing air atatmospheric pressure and or heating in an oven at atmospheric pressureor a vacuum oven.

Pretreatment Conditions

Pretreatment of biomass with the solvent solution comprising one or moresulfide salt under alkaline conditions is carried out in any suitablevessel. Typically the vessel is one that can withstand pressure, has amechanism for heating, and has a mechanism for mixing the contents.Commercially available vessels include, for example, the Zipperclave®reactor (Autoclave Engineers, Erie, Pa.), the Jaygo reactor (JaygoManufacturing, Inc., Mahwah, N.J.), and a steam gun reactor (describedin General Methods Autoclave Engineers, Erie, Pa.). Much larger scalereactors with similar capabilities may be used. Alternatively, thebiomass and organosolv solution may be combined in one vessel, thentransferred to another reactor. Also biomass may be pretreated in onevessel, then further processed in another reactor such as a steam gunreactor (described in General Methods; Autoclave Engineers, Erie, Pa.).

The pretreatment reaction may be performed in any suitable vessel, suchas a batch reactor or a continuous reactor. One skilled in the art willrecognize that at higher temperatures (above 100° C.), a pressure vesselis required. The suitable vessel may be equipped with a means, such asimpellers, for agitating the biomass-organosolv mixture. Reactor designis discussed in Lin, K.-H., and Van Ness, H. C. (in Perry, R. H. andChilton, C. H. (eds), Chemical Engineer's Handbook, 5^(th) Edition(1973) Chapter 4, McGraw-Hill, N.Y.). The pretreatment reaction may becarried out either as a batch, or a continuous process.

Prior to contacting the biomass with solvent, vacuum may be applied tothe vessel containing the biomass. By evacuating air from the pores ofthe biomass, better penetration of the solvent into the biomass may beachieved. The time period for applying vacuum and the amount of negativepressure that is applied to the biomass will depend on the type ofbiomass and can be determined empirically so as to achieve optimalpretreatment of the biomass (as measured by the production offermentable sugars following saccharification).

The heating of the biomass with solvent is carried out at a temperatureof from about 100° C. to about 220° C., about 150° C. to 200° C., orabout 165° C. to about 195° C. The heated solution may be cooled rapidlyto room temperature. In still another embodiment, the heating of thebiomass is carried out at a temperature of about 180° C. Heating of thebiomass-solvent suspension may occur for about 5 minutes to about 5hours, or for about 30 minutes to about 3 hours, or more preferably fromabout 1 to 2 hours.

For the pretreatment methods described herein, the temperature, pH, timeof pretreatment and concentration of reactants such as the organicsolvent and ammonium sulfide solutions, under alkaline conditions, andthe concentration of one or more additional reagents, biomassconcentration, biomass type and biomass particle size are related; thusthese variables may be adjusted as necessary for each type of biomass tooptimize the pretreatment processes described herein.

The pretreatment of biomass with the solvent solution, one or morealkylamine and one or more sulfide salts occurs under alkalineconditions at a pH that is equal to or greater than the pKa of thenucleophiles present. Under these high pH conditions at least 50% of thenucleophiles are in their deprotonated states. Deprotonation typicallyincreases the reactivity of the nucleophiles. The nucleophiles present,in addition to alkylamine, can include ammonia, thiols, polysulfides, orhydrosulfide.

Following pretreatment at elevated temperature the biomass is filteredunder pressure. The filtration may either be preceded or not by cooling.Following filtration, the biomass may be washed one or more times withhydrated organic solvent at elevated or at ambient temperature. It maythen either be washed with water or dried to remove the organic solventand then saccharified. Methods for drying the biomass were describedabove.

To assess performance of the pretreatment, i.e., the production ofreadily saccharifiable carbohydrate-enriched biomass and subsequentsaccharification, separately or together, the theoretical yield ofsugars derivable from the starting biomass can be determined andcompared to measured yields. Pretreatment performance may be furtherassessed by relating how enzyme loadings affect target product yields inoverall system performance.

Further Processing Saccharification

Following pretreatment, the readily saccharifiable carbohydrate-enrichedbiomass comprises a mixture of organic solvent, a sulfide salt and anyadditional components of the solvent solution such as alkylamines orammonia; fragmented and extracted lignin; and polysaccharides. Prior tofurther processing, the ammonium or alkylammonium sulfides orpolysulfides, and/or additional solvent components such as alkylaminesor ammonia and lignin fragments may be removed from the pretreatedbiomass by filtration and washing the sample with EtOH in H₂O (0% to100% EtOH v/v) or water. The biomass may be washed with water to removeEtOH or be dried resulting in carbohydrate-enriched, readilysaccharifiable biomass and the concentration of glucan, xylan andacid-insoluble lignin content of the said biomass may be determinedusing analytical means well known in the art. It is a real benefit ofthis invention that the pretreated biomass can be either washed withwater or dried for saccharification. The readily saccharifiable biomassmay then be further hydrolyzed in the presence of a saccharificationenzyme consortium to release oligosaccharides and/or monosaccharides ina hydrolysate.

Surfactants such as Tween 20 or Tween 80 or polyoxyethylenes such as PEG2000, 4000 or 8000 may be added to improve the saccharification process(U.S. Pat. No. 7,354,743 B2, incorporated herein by reference). Theaddition of surfactant (e.g., Tween 20) to the enzymaticsaccharification often enhances the rate and yield of monomeric sugarrelease. It is likely that the surfactant coats any residual lignin,decreasing the non-productive binding of the enzyme to the lignin. Analternative approach is to either enhance the extraction of lignin inthe pretreatment or to modify the lignin chemically such that lessenzyme is lost to lignin adsorption.

Saccharification enzymes and methods for biomass treatment are reviewedin Lynd, L. R., et al., (Microbiol. Mol. Biol. Rev., 66:506-577, 2002).The saccharification enzyme consortium may comprise one or moreglycosidases; the glycosidases may be selected from the group consistingof cellulose-hydrolyzing glycosidases, hemicellulose-hydrolyzingglycosidases, and starch-hydrolyzing glycosidases. Other enzymes in thesaccharification enzyme consortium may include peptidases, lipases,ligninases and esterases.

The saccharification enzyme consortium comprises one or more enzymesselected primarily, but not exclusively, from the group “glycosidases”which hydrolyze the ether linkages of di-, oligo-, and polysaccharidesand are found in the enzyme classification EC 3.2.1.x (EnzymeNomenclature 1992, Academic Press, San Diego, Calif. with Supplement 1(1993), Supplement 2 (1994), Supplement 3 (1995, Supplement 4 (1997) andSupplement 5 [in Eur. J. Biochem., 223:1-5, 1994; Eur. J. Biochem.,232:1-6, 1995; Eur. J. Biochem., 237:1-5, 1996; Eur. J. Biochem.,250:1-6, 1997; and Eur. J. Biochem., 264:610-650 1999, respectively]) ofthe general group “hydrolases” (EC 3). Glycosidases useful in thepresent method can be categorized by the biomass component that theyhydrolyze. Glycosidases useful for the present method includecellulose-hydrolyzing glycosidases (for example, cellulases,endoglucanases, exoglucanases, cellobiohydrolases, β-glucosidases),hemicellulose-hydrolyzing glycosidases (for example, xylanases,endoxylanases, exoxylanases, β-xylosidases, arabino-xylanases, mannases,galactases, pectinases, glucuronidases), and starch-hydrolyzingglycosidases (for example, amylases, α-amylases, β-amylases,glucoamylases, α-glucosidases, isoamylases). In addition, it may beuseful to add other activities to the saccharification enzyme consortiumsuch as peptidases (EC 3.4.x.y), lipases (EC 3.1.1.x and 3.1.4.x),ligninases (EC 1.11.1.x), and feruloyl esterases (EC 3.1.1.73) to helprelease polysaccharides from other components of the biomass. It is wellknown in the art that microorganisms that producepolysaccharide-hydrolyzing enzymes often exhibit an activity, such ascellulose degradation, that is catalyzed by several enzymes or a groupof enzymes having different substrate specificities. Thus, a “cellulase”from a microorganism may comprise a group of enzymes, all of which maycontribute to the cellulose-degrading activity. Commercial ornon-commercial enzyme preparations, such as cellulase, may comprisenumerous enzymes depending on the purification scheme utilized to obtainthe enzyme. Thus, the saccharification enzyme consortium of the presentmethod may comprise enzyme activity, such as “cellulase”, however it isrecognized that this activity may be catalyzed by more than one enzyme.

Saccharification enzymes may be obtained commercially, in isolated form,such as Spezyme® CP cellulase (Genencor International, Rochester, N.Y.)and Multifect® xylanase (Genencor). In addition, saccharificationenzymes may be expressed in host microorganisms at the biofuels plant,including using recombinant microorganisms.

One skilled in the art would know how to determine the effective amountof enzymes to use in the consortium and adjust conditions for optimalenzyme activity. One skilled in the art would also know how to optimizethe classes of enzyme activities required within the consortium toobtain optimal saccharification of a given pretreatment product underthe selected conditions.

Preferably the saccharification reaction is performed at or near thetemperature and pH optima for the saccharification enzymes. Thetemperature optimum used with the saccharification enzyme consortium inthe present method ranges from about 15° C. to about 100° C. In anotherembodiment, the temperature optimum ranges from about 20° C. to about80° C. and most typically 45-50° C. The pH optimum can range from about2 to about 11. In another embodiment, the pH optimum used with thesaccharification enzyme consortium in the present method ranges fromabout 4 to about 5.5.

The saccharification can be performed for a time of about several min toabout 120 hours, and preferably from about several minutes to about 48hours. The time for the reaction will depend on enzyme concentration andspecific activity, as well as the substrate used, its concentration(i.e., solids loading) and the environmental conditions, such astemperature and pH. One skilled in the art can readily determine optimalconditions of temperature, pH and time to be used with a particularsubstrate and saccharification enzyme(s) consortium.

The saccharification can be performed batch-wise or as a continuousprocess. The saccharification can also be performed in one step, or in anumber of steps. For example, different enzymes required forsaccharification may exhibit different pH or temperature optima. Aprimary treatment can be performed with enzyme(s) at one temperature andpH, followed by secondary or tertiary (or more) treatments withdifferent enzyme(s) at different temperatures and/or pH. In addition,treatment with different enzymes in sequential steps may be at the samepH and/or temperature, or different pHs and temperatures, such as usingcellulases stable and more active at higher pHs and temperaturesfollowed by hemicellulases that are active at lower pHs andtemperatures.

The degree of solubilization of sugars from biomass followingsaccharification can be monitored by measuring the release ofmonosaccharides and oligosaccharides. Methods to measure monosaccharidesand oligosaccharides are well known in the art. For example, theconcentration of reducing sugars can be determined using the1,3-dinitrosalicylic (DNS) acid assay (Miller, G. L., Anal. Chem., 31:426-428, 1959). Alternatively, sugars can be measured by HPLC using anappropriate column as described below.

Fermentation to Target Products

The readily saccharifiable biomass produced by the present methods maybe hydrolyzed by enzymes as described above to produce fermentablesugars which then can be fermented into a target product. “Fermentation”refers to any fermentation process or any process comprising afermentation step. Target products include, without limitation alcohols(e.g., arabinitol, butanol, ethanol, glycerol, methanol,1,3-propanediol, sorbitol, and xylitol); organic acids (e.g., aceticacid, acetonic acid, adipic acid, ascorbic acid, citric acid,2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid,gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid,itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,propionic acid, succinic acid, and xylonic acid); ketones (e.g.,acetone); amino acids (e.g., aspartic acid, glutamic acid, glycine,lysine, serine, and threonine); gases (e.g., methane, hydrogen (H₂),carbon dioxide (CO₂), and carbon monoxide (CO)).

Fermentation processes also include processes used in the consumablealcohol industry (e.g., beer and wine), dairy industry (e.g., fermenteddairy products), leather industry, and tobacco industry.

Further to the above, the sugars produced from saccharifying thepretreated biomass as described herein may be used to produce ingeneral, organic products, chemicals, fuels, commodity and specialtychemicals such as xylose, acetone, acetate, glycine, lysine, organicacids (e.g., lactic acid), 1,3-propanediol, butanediol, glycerol,1,2-ethanediol, furfural, polyhydroxy-alkanoates, cis,cis-muconic acid,and animal feed (Lynd, L. R., Wyman, C. E., and Gerngross, T. U.,Biocom. Eng. Biotechnol. Prog., 15: 777-793, 1999; and Philippidis, G.P., Cellulose bioconversion technology, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212, 1996; and Ryu, D. D. Y., and Mandels, M.,Cellulases: biosynthesis and applications, Enz. Microb. Technol., 2:91-102, 1980).

Potential coproduction of products may also be produced, such asmultiple organic products from fermentable carbohydrate. Lignin-richresidues remaining after pretreatment and fermentation can be convertedto lignin-derived chemicals, chemical building blocks or used for powerproduction.

Conventional methods of fermentation and/or saccharification are knownin the art including, but not limited to, saccharification,fermentation, separate hydrolysis and fermentation (SHF), simultaneoussaccharification and fermentation (SSF), simultaneous saccharificationand cofermentation (SSCF), hybrid hydrolysis and fermentation (HHF), anddirect microbial conversion (DMC).

SHF uses separate process steps to first enzymatically hydrolyzecellulose to sugars such as glucose and xylose and then ferment thesugars to ethanol. In SSF, the enzymatic hydrolysis of cellulose and thefermentation of glucose to ethanol is combined in one step (Philippidis,G. P., supra). SSCF includes the cofermentation of multiple sugars(Sheehan, J., and Himmel, M., Bioethanol, Biotechnol. Prog., 15:817-827, 1999). HHF includes two separate steps carried out in the samereactor but at different temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(cellulase production, cellulose hydrolysis, and fermentation) in onestep (Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I. S.,Microbiol. Mol. Biol. Rev., 66: 506-577, 2002). These processes may beused to produce target products from the readily saccharifiable biomassproduced by the pretreatment methods described herein.

Advantages of the Present Methods

Sulfides are among the best soft nucleophiles. By incorporating sulfidesinto an organic solvent process under alkaline conditions (due to thepresence of alkali metal or alkaline earth hydroxide or ammonia oralkylamine or a combination thereof), the anionic sulfide(hydrosulfide), is primed to carry out substitution reactions on thearyl ethers of the lignin. The alkaline conditions also favor theformation of quinone methides from lignin. These are also readilyattacked by sulfides. The presence of ammonia and/or alkylamines, inaddition to raising the pH, supplements the sulfide nucleophilicchemistry in attacking the lignin, and likely protect the polysaccharideagainst peeling reactions, that result in monosaccharide release andloss at high pH. The use of alkylamines and/or ammonia as bases avoidsthe generation of an inorganic waste stream which would otherwise add tothe financial and environmental cost of the process. The sulfides canalso act as a reducing agents, promoting the reduction of quinonemethides, eliminating 3-aryl ethers as phenoxyl radicals and protectingsugar residues from oxidative reactions. The use of sulfides in thelignocellulosic biomass pretreatment process enhances ligninfragmentation and therefore increases the selectivity of ligninextraction with respect to carbohydrate, producing carbohydrate-enrichedbiomass that is highly susceptible to enzymatic saccharification.Methods described in this invention for pretreatment of thelignocellulosic biomass using an organic solvent-mediated fragmentationin the presence of one or more ammonium or alkylammonium sulfides andvarious nucleophiles in combination with selective extraction of ligninat elevated temperatures under alkaline conditions will provide acost-effective process to obtain carbohydrate-enriched biomass forenzymatic saccharification. Such biomass then, produces very high yieldsof fermentable sugars (glucose, as well as xylose) for theirbioconversion to value-added chemicals and fuels.

EXAMPLES Pretreatment of Biomass to Obtain Readily SaccharifiableCarbohydrate-Enriched Biomass

The goal of the experimental work described below was to develop apretreatment process for lignocellulose that maximized both ligninextraction and sugar retention and to produce a readily saccharifiablecarbohydrate-enriched biomass that may be further processed to result ina maximal monomeric sugar yield following enzymatic saccharification.The approach adopted was to selectively fragment and extract the lignininto a suitable solvent while retaining the sugars in the solidsresidue. The following experiments show the development of a solventsolution that combines the presence of nucleophiles like sulfide salts,alkylamines, NH₃, and thiol for selective extraction of lignin. It wasfound that the combined presence of an organic solvent and a sulfidesalt and optionally certain nucleophiles like alkylamine, NH₃, and thiolreactants selectively fragmented and dissolved the lignin components ofbiomass providing for the generation of readily saccharifiablecarbohydrate-enriched biomass.

Ground sugar cane bagasse, which was milled in a Wiley Knife mill,through a 1 mm sieve, was used in all Examples.

The following abbreviations are used in the Examples: “HPLC” is HighPerformance Liquid Chromatography, “C” is degrees Centigrade or Celsius;“%” is percent; “wt” is weight; “w/w” is weight for weight; “mL” ismilliliter; “OD” is outer diameter; “ID” is internal diameter; “h” ishour(s); “rpm” is revolution per minute; “EtOH” is ethanol; “mg/g” ismilligram per gram; “g/100 mL” is gram per 100 milliliters; “N” isnormal; “g” is gram; “w/v” is weight per volume; “v/v” is volume forvolume; “mm” is millimeter; “mL/min” is milliliter per minute; “min” isminutes; “mM” is millimolar.

Materials

Sulfuric acid, ammonium hydroxide, acetic acid, acetamide, yeastextract, 2-morpholinoethanesulfonic acid (MES), potassium phosphate,glucose, xylose, tryptone, sodium chloride and citric acid, monomethyland dimethylamine were obtained from Sigma-Aldrich (St. Louis, Mo.).Spezyme CP and Multifect CX12L were from Genecor (GenencorInternational, Palo Alto, Calif.) and Novozyme 188 was from Novozyme(Bagsvaerd, Denmark).

Example 1 Effective Ethanol Concentration

The purpose of this Example was to examine the effect of theconcentration of solvent (e.g., ethanol) in water on the recovery ofcarbohydrate and on the solubilization/extraction of lignin in theabsence of pH control. Bagasse (0.2 g, 95.78% dry matter) was suspendedin 1.56 mL of an EtOH in water solution containing variousconcentrations (from 0 to 80%) of EtOH. The suspensions were loaded intotype 316 stainless steel tubing (¼ inches ID, ⅜ inches OD, 4 incheslong) capped by Swagelock fittings (Penn Fluid System Technologies,Huntingdon Valley, Pa.). These were placed in a fluidized sand bath(Techne Model SBS-4, Techne Inc., Burlington, N.J.) and heated at 180°C. for 2 h and cooled rapidly by plunging into a water bath at roomtemperature. The samples were removed from the tubes and filtered bycentrifugation at 14,000 rpm using Spin-X filters (Costar, Corning Inc.,Corning N.Y.) at room temperature in a table top centrifuge (Spectrifuge16M, Labnet International Inc., Edison, N.J.) to remove the dissolvedlignin. The retentate of each sample was washed (4×) with 0.5 mL of EtOHin H₂O using the same EtOH concentration as used in the 180° C.treatment (0-80% EtOH in H₂O). The samples were then allowed to air dryat room temperature (to ˜92% dry matter) and the glucan, xylan andacid-insoluble lignin contents of the residues determined using theNational Renewable Energy Laboratory (NREL) procedure (Determination ofStructural Carbohydrates and Lignin in Biomass—Version 2006, ArnieSluiter et al., available from the NREL website.

Subsequent Enzymatic Saccharification

The air-dried sample prepared above was suspended in 50 mM citratebuffer, pH 4.6 at a ˜14% solids loading. The saccharification enzymes,e.g. Spezyme CP, Multifect CX12L and Novozyme 188 were added atconcentrations of 6:3:6 mg/g cellulose, respectively. Also added were 1%(g/100 mL) Tween 20 and 0.01% (w/v) NaN₃, the latter to preventmicrobial growth. Samples (˜0.4 mL) were placed in screw cap vialscontaining two 5 mm glass beads and incubated at 46° C. on a rotaryshaker run at 250 rpm. Aliquots were removed for analysis at 4 h and atevery 24 h interval from the start and diluted 41.25-fold with 0.01 NH₂SO₄. The samples were then filtered through Spin-X filters and thefiltrates were analyzed by HPLC (Agilent series 1100/1200, AgilentTechnologies, Wilmington, Del.). A BioRad HPX-87H Aminex column (Bio-RadLaboratories, Hercules, Calif.) was used to fractionate the releasedsugars using 0.01 N H₂SO₄ as the mobile phase at a flow rate of 0.6mL/min. The column was maintained at 60° C. A differential refractiveindex detector was used to detect the eluted sugars and was maintainedat 55° C. The retention times for glucose, xylose and arabinose were9.05, 9.72 and 10.63 min, respectively. Table 1A outlines thepercentages of glucan and xylan recovery and the percent change in acidinsoluble (AI) lignin content after pretreatments at EtOH concentrationsof 0%-80%. Concentration of Bagasse was (0.2 g/1.56 mM) variableconcentrations of EtOH were used at 180° C. for 2 h

TABLE 1A Glucan and xylan recovery following pretreatment according toExample 1 Pretreatment % Glucan % Xylan Al lignin (% EtOH in recovery inrecovery in content water) residue residue % change 0 83.0% 29.0% +27.6%20 88.7% 30.8% +15.2% 40 86.0% 57.6%   −10% 60 91.9% 87.4% −25.6% 8088.6% 91.1% −28.8%

Results shown in Table 1A indicate that lignin extraction increased withincreasing EtOH content presumably because the solubility of ligninincreased with increasing EtOH concentration. However, the amount oflignin extracted remained modest even at high ethanol concentrations.

Hemicellulose hydrolysis and the solubility of xylose oligomersdecreases with increasing EtOH, increasing the recovery of xylan andxylose oligomers in the residue. The amount of acetate liberated by thepretreatment also decreased with increasing EtOH content, consistentwith decreasing auto hydrolysis of the biomass at increasing EtOHconcentration.

Table 1B shows the glucose and xylose yields after 96 h of enzymaticsaccharification following pretreatment at different EtOHconcentrations. The saccharification of cellulose increased when theconcentration of EtOH in pretreatment was increased from 0 to 20%, butthen declined with higher pretreatment concentrations of EtOH. A likelydecrease in partial hydrolysis of lignin and cellulose (increase indegree of polymerization, of cellulose which lowered the glucose yieldon subsequent saccharification—Table 1B) was observed at concentrationsof more than 20% EtOH.

TABLE 1B Monomeric glucose and xylose yields following enzymaticsaccharification for 96 h, pretreated as described in Example 1 GlucoseXylose Glucose Xylose monomer monomer monomer monomer saccharificationsaccharification overall overall % EtOH only only yield (% yield (% inwater (% theoretical (% theoretical theoretical theoretical (v/v) yield)yield) yield) yield) 0 38.43 34.98 31.86 10.16 20 44.48 45.52 39.4614.01 40 29.62 38.55 25.45 22.23 60 16.81 24.64 15.45 21.52 80 6.8 7.226.02 7.01

The monomeric sugar recoveries (Table 1B), particularly of xylose, werequite poor at the lower EtOH concentrations. At low EtOH concentration,the acidic conditions, produced at high temperatures by hydrolysis ofthe acetyl groups of the hemicellulose, hydrolyze the hemicellulose. Thesolubilized xylose and some glucose is lost in the filtration and washesthat follow the pretreatment. At higher EtOH concentrations there isless partial hydrolysis of the cellulose, hemicellulose and lignin whichlowers the saccharification yield. The behavior at the low and highethanol concentrations together produce low overall yields of monomericglucose and xylose.

Example 2 Effect of Alkaline Organic Solvent Solution Pretreatment onLignin Extraction

The purpose of this Example was to examine the effect of raising the pHof an organic solvent solution pretreatment at different EtOH in H₂Oratios on carbohydrate retention and lignin extraction and on monomericsugar during subsequent enzymatic saccharification. Given thatautohydrolysis lowers the pH, hydrolyzes xylan, and promotes the loss ofxylose, the pH of the pretreatment was elevated by the addition of NaOH.The effect of higher pH on xylose recovery is demonstrated below. Sugarcane bagasse (0.25 g, 95.78% dry matter) was suspended in 1.75 mL of asolvent containing EtOH (20-80% in water) and 8% NaOH (w/w biomass) plus1 mg anthraquinone (AQ, a catalyst for lignin fragmentation). Theinitial pH of this solution was ˜13.7. As described in Example 1, thesuspensions were loaded into type 316 stainless steel tubing, capped,treated at 168° C. for 140 min and cooled in room-temperature water. Thesamples were removed from the pressure vessels, filtered, washed,air-dried and analyzed all as described above in Example 1. The glucan,xylan, arabinan contents and change in lignin content followingpretreatment are shown in Table 2A.

Subsequent enzymatic saccharification was carried out as described inExample 1 except that the Spezyme:Multifect:Novozymes 188 ratio was12:6:1.2 mg/g dry solids in the presence of 1% Tween 20 (w/v). Table 2Bshows the monomeric sugar yields after 96 h of enzymaticsaccharification of biomass previously pretreated at the different EtOHconcentrations.

TABLE 2A Glucan, xylan and arabinan yields following pretreatmentaccording to Example 2 Pretreatment % Glucan % Xylan % Arabinan Allignin % EtOH in recovery recovery in recovery in content water inresidue residue residue % change 20 77.5% 74.6% 51.3% −48 45 84.0% 85.1%68.0% −64 60 83.6% 85.5% 76.0% −63 70 81.3% 84.2% 75.8% −65 80 80.0%84.2% 86.6% −50

TABLE 2B Monomeric glucose and xylose yields following enzymaticsaccharification for 96 h, pretreated as described in Example 2 GlucoseGlucose Xylose monomer Xylan monomer monomer monomer saccharificationsaccharification overall overall only only yield (% yield (% % EtOH (%theoretical (% theoretical theoretical theoretical in H₂O yield) yield)yield) yield) 20 57.72 68.56 44.7 51.2 45 58.19 73.08 48.9 62.2 60 49.5164.56 41.4 55.2 70 24.48 39.06 19.9 32.9 80 0.63 1.33 0.5 1.1

As can be seen in Tables 2A and 2B, the alkaline conditions of thisexperiment substantially increased the retention of xylan in thepretreatment compared to the autohydrolysis experiments of Example 1.This effect was most pronounced at low EtOH concentrations. The NaOHprevented the solution from becoming acidic (final pH ˜10.7) andtherefore protected the hemicellulose from acid-catalyzed hydrolysis. Inaddition, significantly more lignin was extracted, presumably throughbase catalyzed fractionation of the lignin. The overall monomeric sugaryields following saccharification were substantially higher than thoseobserved in Example 1. The higher sugar recovery and the greater ligninextraction in the pretreatment, increased the yields of the subsequentenzymatic saccharification. The xylose and glucose saccharificationyields peaked at ˜45% EtOH as a consequence of two opposing processes,i.e., the increasing extraction of lignin at higher EtOH which tends toincrease the sugar yields, and the decreasing partial hydrolysis ofhemicellulose and of lignin as the EtOH concentration is furtherincreased. It is likely that the formation of quinone methides, whichcould repolymerize or react with sugars, and “peeling’ and alkalinescission reactions of polysaccharide all together contribute to limitthe overall sugar yields.

Example 3 Pretreatment of Biomass Using Ammonium Sulfide During LigninExtraction

The purpose of this Example was to study the effect of ammonium sulfideon biomass pretreatment. Pretreatment was performed as in Example 1except that sugar cane bagasse (0.375 g, 95.78% dry matter) wassuspended in 1.125 mL of solvent (70% EtOH in H₂O (v/v)) containing 14%MA (w/w biomass) plus 2% or 6% (NH₄)₂S (w/w biomass). The suspensionswere loaded into type 316 stainless steel pressure vessels ( 3/16 inchesID, ¼ inches OD, 4 inches long), capped and treated as described abovein Example 1, except that solids loading was higher and the samples wereheated at 168° C., for 140 min. The subsequent enzymaticsaccharification was performed for 96 h as described in Example 1 exceptthat the Spezyme:Multifect:Novozymes 188 ratio was 6.68:3.34:1.67 mg/gdry solids in the presence and absence of 1% Tween 20 (w/v) at a solidsloading of 14% (w/w). The saccharification yields on material pretreatedas described in the presence and absence of 2% and 6% ammonium sulfide(w/w biomass) are shown in Table 3.

TABLE 3 Yields of glucan and xylan following pretreatment according toExample 3 Glucose Xylose Glucose Xylose monomer monomer monomer monomerSample sacch. sacch. sacch. sacch. 70% EtOH only only only (% only (% inH₂O % Glucan % Xylan (% (% theoretical theoretical (v/v) plus recoveryrecovery theoretical theoretical yield) yield) in w/w of in in yield)yield) with with biomass solids solids no Tween no Tween Tween Tween 14%MA 90.60 97.52 69.07 58.26 75.96 67.5 14% MA + 91.62 98.03 78.9 68.6884.79 76.39 2% (NH₄)₂S 14% MA + 87.02 92.43 84.2 73 90.87 83.23 6%(NH₄)₂S

The comparison of the enzymatic saccharifications in the absence ofTween 20 following pretreatment with 70% EtOH in H₂O (v/v) plus 14% MA(w/w biomass) containing either 0%, 2% or 6% (NH₄)₂S (w/w biomass)showed that (NH₄)₂S when present in the pretreatment promoted subsequentenzymatic saccharification. The enhanced saccharification was likelyassociated with an increased fragmentation and extraction of the lignin(FIG. 1), through reactions similar to those that occur withthioglycolic acid. Table 3 shows that the effect of the 2% (NH₄)₂Spretreatment was quite marked for the saccharification of xylan toxylose and for glucan to glucose. The HPLC chromatographic profiles ofthe hydrolysates indicated, as in the case of thioglycolic acid, thatthe enhanced extraction of the lignin by (NH₄)₂S in the pretreatmentwith no surfactant in the saccharification behaved similarly to theaddition of surfactant in the saccharification but without (NH₄)₂S inthe pretreatment. The surfactant coats the residual lignin while the(NH₄)₂S reduces the amount of residual lignin. The presence of 6%(NH₄)₂S to the pretreatment produces an additional boost in thesaccharification yield, well above that of the sample saccharified withTween 20, but pretreated in the absence of (NH₄)₂S.

Example 4 Ammonium Sulfide Enhanced Lignin Extraction

Pretreatment was performed as in Example 3 except that the 70% EtOH inH₂O (v/v) solvent in which the bagasse was suspended contained 14% MA(w/w biomass) with no additions and with 2% or 6% (NH₄)₂S (w/w biomass).The samples were heated at 187° C. for 1 h. FIG. 1 shows the UVabsorbance spectra of the filtrates following pretreatment, diluted5000-fold with 70% EtOH in H₂O (v/v). The addition of 2% and 6% (NH₄)₂Sto the 70% EtOH/H₂O plus MA showed a very large enhancement in the UVabsorption of the filtrate following pretreatment, indicating anincrease in the extracted lignin. The enhancement of the ligninextraction by inclusion of (NH₄)₂S in the pretreatment is consistentwith the significant enhancement of the subsequent enzymaticsaccharification (Table 3).

1. A method for producing carbohydrate-enriched biomass comprising: (a)providing lignocellulosic biomass comprising lignin; (b) suspending thebiomass of (a) in an organic solvent solution comprising water and oneor more sulfide salt under alkaline conditions whereby a biomass-solventsuspension is formed; (c) heating the biomass-solvent suspension to atemperature of about 100° C. to about 220° C. for about 5 minutes toabout 5 hours whereby lignin is fragmented and is dissolved in thesuspension; and (d) filtering free liquid whereby the dissolved ligninis removed and whereby readily carbohydrate-enriched biomass isproduced.
 2. The method of claim 1 further comprising: (e) washing thebiomass produced in step (d) with a solvent solution.
 3. The method ofclaim 2, further comprising: (f) washing the biomass produced in step(e) with water to produce readily saccharifiable carbohydrate-enrichedbiomass.
 4. The method of claim 2 further comprising drying biomassproduced in step (e) to produce readily saccharifiablecarbohydrate-enriched biomass.
 5. The method of claim 2 or 3, furthercomprising repeating steps (e) and (f) one or more times.
 6. The methodof claim 1 wherein the heating step of (c) occurs in a sealed pressurevessel.
 7. The method of claim 1 wherein the filtering step of (d)occurs under pressure.
 8. The method of claim 1 wherein the organicsolvent solution further comprises an additional nucleophile selectedfrom the group consisting of NH₃, one or more alkylamines, NaOH,polysulfide, hydropolysulfide reagents, and combinations thereof.
 9. Themethod of claim 8 wherein the additional nucleophile is one or morealkylamine and said one or more alkylamine is at a concentration ofabout up to 20% by weight of dry biomass
 10. The method of claim 1wherein the solvent solution to biomass in step (b) has a weight ratioof about 10 to 1 to 0.5 to
 1. 11. The method of claim 1, wherein theheated suspension of step (c) is cooled to room temperature beforefiltering in step (d).
 12. The method of claim 2 further comprisingevaporating off the solvent under vacuum of the filtered and washedbiomass after step (e).
 13. The method of claim 3, 4 or 12, furthercomprising saccharifying the biomass with an enzyme consortium wherebyfermentable sugars are produced.
 14. The method of claim 3, furthercomprising saccharifying the biomass without drying by contacting saidbiomass with an enzyme consortium after washing in step (f), wherebyfermentable sugars are produced.
 15. The method of claim 13 or 14,further comprising fermenting the sugars to produce a target product.16. The method of claim 15 wherein the target product is selected fromthe group consisting of alcohols, organic acids, amino acids and gases.17. The method of claim 1 wherein the biomass is selected from the groupconsisting of switchgrass, waste paper, sludge from paper manufacture,corn fiber, corn cobs, corn husks, corn stover, grasses, wheat, wheatstraw, hay, barley, barley straw, rice straw, sugar cane bagasse, sugarcane straw, yellow poplar, sorghum, soy, components obtained fromprocessing of grains, trees, branches, roots, leaves, wood chips,sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manureand combinations thereof.
 18. A method of simultaneous fragmentation andselective extraction of lignin from lignocellulosic biomass to produceda substantially lignin-free biomass comprising: (a) providing: 1) anamount of lignocellulosic biomass; 2) a multi-component organic solventsolution comprising from about 40% to about 70% ethanol in water; 3) oneor more sulfide salt(s); and 4) one or more alkylamine(s) under alkalineconditions; (b) contacting said biomass with the multi-component solventsolution of (a) whereby a solvent-biomass mixture is produced; (c)placing the solvent-biomass mixture in a sealed pressure vessel wherebythe mixture of (b) is heated from about 100° C. to about 220° C. forabout 5 minutes to about 5 hours whereby lignin is fragmented anddissolved in the solvent; (d) removing the dissolved lignin of (c) byfiltration; and (e) washing the residual biomass with organic solvent,whereby substantially lignin-free biomass is produced.
 19. The method ofclaim 18 wherein the solvent at step (e) may contain water
 20. Themethod of claim 18 wherein the substantially lignin-free biomass is fromabout 60% to about 100% original weight of the biomass.
 21. The methodof any one of claim 1 or 18, wherein the organic solvent solutionfurther comprises one or additional component selected from the groupconsisting of alkali or alkaline earth hydroxides or carbonates,ammonia, thiols, polysulfides, hydropolysulfides and combinationsthereof.
 22. The method of claim 1 or 18 wherein the solvent solution,and any unreacted sulfide salts or other unreacted components arerecyclable.
 23. The method of claim 1 or 18 wherein said organic solventsolution comprises a solvent selected from the group consisting ofalcohols, diols and aprotic solvents
 24. The method of claim 23 whereinthe organic solvent solution comprises a solvent selected from the groupconsisting of methanol, ethanol, propanol, butanol, pentanol andhexanol, isomers thereof, and diols thereof.
 25. The method of claim 1or 18 wherein the lignocellulosic biomass of step (a) has a carbohydratecontent that is highly conserved through steps (a) through (d).
 26. Themethod of claim 8 or 18 wherein the one or more alkylamines is selectedfrom the group consisting of R—NH₂, R₂—NH, R₃N, (H₂N—R—NH₂),(H₂N—R(NH₂)₂), (HO—R—NH₂), ((HO)₂—R—NH₂), (HO—R—(NH₂)₂), (HS—R—NH₂),((HS)₂—R—NH₂), (HS—R—(NH₂)₂) and (H₂N—R(OH)(SH) and combinationsthereof, wherein R is independently a monovalent, divalent or trivalent1-6 carbon alkane, alkene or alkyne, linear, cyclic or branched.
 27. Themethod of claim 26 wherein R is independently methyl, ethyl, propyl orbutyl.
 28. The method of claim 26 wherein the alkylamine is methylamine.29. The method of claim 1 or 18 where the sulfide salt is selected fromthe group consisting of alkali metal and alkaline earth sulfides (e.g.Na₂S), alkali metal and alkaline earth hydrosulfides (e.g. NaHS), alkalimetal and alkaline earth polysulfides (e.g., Na₂S_(n)), alkali metal andalkaline earth hydropolysulfides (e.g., NaHS_(n)), ammonium sulfide,hydrosulfide, polysulfides, and hydropolysulfides, alkylammoniumsulfide, hydrosulfide, polysulfides, and hydropolysulfides.
 30. Themethod of claims 1 and 18 wherein the temperature of step (c) is fromabout 165° C. to about 195° C.